One of the requirements of quantitative PCR is to accurately determine the number of copies of starting template in a given sample, this is generally achieved by measuring the cycle threshold (CT; that is, the number of cycles for measured fluorescence to increase above a predetermined threshold) of the amplification of the unknown and referencing this back to a standard curve of known copy number. A lower CT indicates a greater amount of starting template. Typically the CT is determined as the point where the amplification reaction shifts from the baseline to the exponential amplification stage.
Use of a standard curve is however difficult with diagnostic instruments, where it is generally inconvenient to run such a curve. Further, standard curves can differ from reaction to reaction, depending on the reaction conditions, the presence of certain ions in the sample, etc, so it is considered best practice to use a separate standard curve derived for each reaction. This clearly is impractical for diagnostic assays and more specifically in point of care devices that generally rely on a single reaction.
It is known to use a well-characterized amplification internal to the assay amplification, to measure deviation from known metrics; this is also based on CT measurement from real time data. However, this is not always convenient because the internal control can compete for reaction components, reducing the limit of detection and sacrificing sensitivity.
There is a need for an improved quantification method that is compatible with diagnostic instrumentation. We describe a process that provides quantification metrics as part of the same amplification reaction, which also allows use of melt curve analysis to reduce the complexity of the reaction, providing both quantitative and genotyping information from a single amplification.